Generation of plants with improved pathogen resistance

ABSTRACT

The present invention is directed to plants that display a pathogen resistance phenotype due to altered expression of a PPR1 nucleic acid. The invention is further directed to methods of generating plants with a pathogen resistance phenotype.

CROSS REFERENCE TO RELATED APPLICATIONS

This is a divisional application of U.S. patent application Ser. No.10/509,669, filed Mar. 2, 2006, which is the US National Stage ofPCT/US2003/09485, filed on Mar. 27, 2003, which claims the benefit ofU.S. Provisional Application No. 60/368,613, filed Mar. 27, 2002. Theentire disclosures of each of these applications are hereby expresslyincorporated by reference.

BACKGROUND OF THE INVENTION

The control of infection by plant pathogens, which can inhibitproduction of fruits, seeds, foliage and flowers and cause reductions inthe quality and quantity of the harvested crops, is of significanteconomic importance. Pathogens annually cause billions of dollars indamage to crops worldwide (Baker et al. 1997, Science 276:726-733).Consequently, an increasing amount of research has been dedicated todeveloping novel methods for controlling plant diseases. Such studieshave centered on the plant's innate ability to resist pathogen invasionin an effort to buttress the plant's own defenses to counter pathogenattacks (Staskawicz et al. 1995, Science 268:661-667; Baker et al.supra).

Although most crops are treated with agricultural anti-fungal,anti-bacterial agents and/or pesticidal agents, damage from pathogenicinfection still results in revenue losses to the agricultural industryon a regular basis. Furthermore, many of the agents used to control suchinfection or infestation cause adverse side effects to the plant and/orto the environment. Plants with enhanced resistance to infection bypathogens would decrease or eliminate the need for application ofchemical anti-fungal, anti-bacterial and/or pesticidal agents.

There has been significant interest in developing transgenic plants thatshow increased resistance to a broad range of pathogens (Stuiver andCusters 2001, Nature 411:865-8; Melchers and Stuiver 2000, Curr OpinPlant Biol 3:147-52; Rommens and Kishore 2000, Curr Opin Biotechnol 11:120-5; Mourgues et al. 1998, Trends Biotechnol 16:203-10). Theinteraction between Arabidopsis and the oomycete Peronospora parasitica(downy mildew) provides an attractive model system to identify molecularcomponents of the host that are required for recognition of the fungalparasite (Parker et al. 1996, Plant Cell 8:2033-46). A number of geneswhose mis-expression is associated with altered resistance to P.parasitica, as well as other pathogens, have been identified inArabidopsis. Overexpression of the NPR1 gene confers resistance toinfection by P. parasitica as well as the bacterial pathogen Pseudomonassyringae (Cao et al. 1998 Proc Natl Acad Sci USA 95:6531-6536). CPR6 issemi-dominant mutation implicated in multiple defense pathways (Clarkeet al. 1998, Plant Cell 10:557-569). Lsd6 and Lsd7 are dominantmutations that confer heightened disease and result in the developmentof spontaneous necrotic lesions and elevated levels of salicylic acid(Weymann et al 1995, Plant Cell 7:2013-2022). A number of recessivemutations confer P. parasitica resistance, including ssi2, in the SSI2gene encoding a stearoyl-ACP desaturase (Kachroo et al. 2001, Proc NatlAcad Sci USA 98:9448-9453), mpk4, in a MAP kinase gene (Petersen et al.2000, Cell 103:1111-20), and pmr4 (Vogel and Somerville 2000, Proc NatlAcad Sci USA 97:1897-1902). The recessive mutations cpr5 and cpr1 alsoconfer resistance to P. syringae and cause a dwarf phenotype (Bowling etal. 1997, Plant Cell 9:1573-1584; Bowling et al. 1994, Plant Cell6:1845-1857).

Activation tagging in plants refers to a method of generating randommutations by insertion of a heterologous nucleic acid constructcomprising regulatory sequences (e.g., an enhancer) into a plant genome.The regulatory sequences can act to enhance transcription of one or morenative plant genes; accordingly, activation tagging is a fruitful methodfor generating gain-of-function, generally dominant mutants (see, e.g.,Hayashi et al. Science 1992, 258: 1350-1353; Weigel et al. 2000, PlantPhysiology 122:1003-1013). The inserted construct provides a moleculartag for rapid identification of the native plant whose mis-expressioncauses the mutant phenotype. Activation tagging may also causeloss-of-function phenotypes. The insertion may result in disruption of anative plant gene, in which case the phenotype is generally recessive.

Activation tagging has been used in various species, including tobaccoand Arabidopsis, to identify many different kinds of mutant phenotypesand the genes associated with these phenotypes (Wilson et al. 1996,Plant Cell 8:659-671, Schaffer et al. 1998, Cell 93: 1219-1229; Fridborget al. 1999, Plant Cell 11: 1019-1032; Kardailsky et al. 1999, Science286:1962-1965); Christensen et al., 9^(th) International Conference onArabidopsis Research. Univ. of Wisconsin-Madison, Jun. 24-28, 1998,Abstract 165). In one example, activation tagging was used to identifymutants with altered disease resistance (Weigel et al., supra).

SUMMARY OF THE INVENTION

The invention provides a transgenic plant comprising a planttransformation vector comprising a nucleotide sequence that encodes oris complementary to a sequence that encodes a PPR1 polypeptide or anortholog thereof. The transgenic plant is characterized by havingincreased resistance to pathogens controlled by the salicylicacid-dependent resistance pathway relative to control plants.

The present invention further provides a method of producing an alteredpathogen resistance phenotype in a plant. The method comprisesintroducing into plant progenitor cells a vector comprising a nucleotidesequence that encodes or is complementary to a sequence encoding a PPR1polypeptide or an ortholog thereof and growing a transgenic plant thatexpresses the nucleotide sequence. In one embodiment, the PPR1polypeptide has at least 50% sequence identity to the amino acidsequence presented in SEQ ID NO:2 and comprises an AP2 domain. In otherembodiments, the PPR1 polypeptide has at least 80% or 90% sequenceidentity to or has the amino acid sequence presented in SEQ ID NO:2.

The invention further provides plants and plant parts obtained by themethods described herein.

DETAILED DESCRIPTION OF THE INVENTION Definitions

Unless otherwise indicated, all technical and scientific terms usedherein have the same meaning as they would to one skilled in the art ofthe present invention. Practitioners are particularly directed toSambrook et al., Molecular Cloning: A Laboratory Manual (SecondEdition), Cold Spring Harbor Press, Plainview, N.Y., 1989, and Ausubel FM et al., Current Protocols in Molecular Biology, John Wiley & Sons, NewYork, N.Y., 1993, for definitions and terms of the art. It is to beunderstood that this invention is not limited to the particularmethodology, protocols, and reagents described, as these may vary.

As used herein, the term “vector” refers to a nucleic acid constructdesigned for transfer between different host cells. An “expressionvector” refers to a vector that has the ability to incorporate andexpress heterologous DNA fragments in a foreign cell. Many prokaryoticand eukaryotic expression vectors are commercially available. Selectionof appropriate expression vectors is within the knowledge of thosehaving skill in the art.

A “heterologous” nucleic acid construct or sequence has a portion of thesequence that is not native to the plant cell in which it is expressed.Heterologous, with respect to a control sequence refers to a controlsequence (i.e. promoter or enhancer) that does not function in nature toregulate the same gene the expression of which it is currentlyregulating. Generally, heterologous nucleic acid sequences are notendogenous to the cell or part of the genome in which they are present,and have been added to the cell, by infection, transfection,microinjection, electroporation, or the like. A “heterologous” nucleicacid construct may contain a control sequence/DNA coding sequencecombination that is the same as, or different from a controlsequence/DNA coding sequence combination found in the native plant.

As used herein, the term “gene” means the segment of DNA involved inproducing a polypeptide chain, which may or may not include regionspreceding and following the coding region, e.g. 5′ untranslated (5′ UTR)or “leader” sequences and 3′ UTR or “trailer” sequences, as well asintervening sequences (introns) between individual coding segments(exons) and non-transcribed regulatory sequence.

As used herein, “recombinant” includes reference to a cell or vector,that has been modified by the introduction of a heterologous nucleicacid sequence or that the cell is derived from a cell so modified. Thus,for example, recombinant cells express genes that are not found inidentical form within the native (non-recombinant) form of the cell orexpress native genes that are otherwise abnormally expressed, underexpressed or not expressed at all as a result of deliberate humanintervention.

As used herein, the term “gene expression” refers to the process bywhich a polypeptide is produced based on the nucleic acid sequence of agene. The process includes both transcription and translation;accordingly, “expression” may refer to either a polynucleotide orpolypeptide sequence, or both. Sometimes, expression of a polynucleotidesequence will not lead to protein translation. “Over-expression” refersto increased expression of a polynucleotide and/or polypeptide sequencerelative to its expression in a wild-type (or other reference [e.g.,non-transgenic]) plant and may relate to a naturally-occurring ornon-naturally occurring sequence. “Ectopic expression” refers toexpression at a time, place, and/or increased level that does notnaturally occur in the nonaltered or wild-type plant. “Under-expression”refers to decreased expression of a polynucleotide and/or polypeptidesequence, generally of an endogenous gene, relative to its expression ina wild-type plant. The terms “mis-expression” and “altered expression”encompass over-expression, under-expression, and ectopic expression.

The term “introduced” in the context of inserting a nucleic acidsequence into a cell, means “transfection”, or “transformation” or“transduction” and includes reference to the incorporation of a nucleicacid sequence into a eukaryotic or prokaryotic cell where the nucleicacid sequence may be incorporated into the genome of the cell (forexample, chromosome, plasmid, plastid, or mitochondrial DNA), convertedinto an autonomous replicon, or transiently expressed (for example,transfected mRNA).

As used herein, a “plant cell” refers to any cell derived from a plant,including cells from undifferentiated tissue (e.g., callus) as well asplant seeds, pollen, progagules and embryos.

As used herein, the terms “native” and “wild-type” relative to a givenplant trait or phenotype refers to the form in which that trait orphenotype is found in the same variety of plant in nature.

As used herein, the term “modified” regarding a plant trait, refers to achange in the phenotype of a transgenic plant relative to the similarnon-transgenic plant. An “interesting phenotype (trait)” with referenceto a transgenic plant refers to an observable or measurable phenotypedemonstrated by a T1 and/or subsequent generation plant, which is notdisplayed by the corresponding non-transgenic (i.e., a genotypicallysimilar plant that has been raised or assayed under similar conditions).An interesting phenotype may represent an improvement in the plant ormay provide a means to produce improvements in other plants. An“improvement” is a feature that may enhance the utility of a plantspecies or variety by providing the plant with a unique and/or novelquality.

An “altered pathogen resistance phenotype” refers to detectable changein the response of a genetically modified plant to pathogenic infection,compared to the similar, but non-modified plant. The phenotype may beapparent in the plant itself (e.g., in growth, viability or particulartissue morphology of the plant) or may be apparent in the ability of thepathogen to proliferate on and/or infect the plant. As used herein,“improved pathogen resistance” refers to increased resistance to apathogen.

As used herein, a “mutant” polynucleotide sequence or gene differs fromthe corresponding wild type polynucleotide sequence or gene either interms of sequence or expression, where the difference contributes to amodified plant phenotype or trait. Relative to a plant or plant line,the term “mutant” refers to a plant or plant line which has a modifiedplant phenotype or trait, where the modified phenotype or trait isassociated with the modified expression of a wild type polynucleotidesequence or gene.

As used herein, the term “T1” refers to the generation of plants fromthe seed of T0 plants. The T1 generation is the first set of transformedplants that can be selected by application of a selection agent, e.g.,an antibiotic or herbicide, for which the transgenic plant contains thecorresponding resistance gene. The term “T2” refers to the generation ofplants by self-fertilization of the flowers of T1 plants, previouslyselected as being transgenic.

As used herein, the term “plant part” includes any plant organ ortissue, including, without limitation, seeds, embryos, meristematicregions, callus tissue, leaves, roots, shoots, gametophytes,sporophytes, pollen, and microspores. Plant cells can be obtained fromany plant organ or tissue and cultures prepared therefrom. The class ofplants which can be used in the methods of the present invention isgenerally as broad as the class of higher plants amenable totransformation techniques, including both monocotyledenous anddicotyledenous plants.

As used herein, “transgenic plant” includes reference to a plant thatcomprises within its genome a heterologous polynucleotide. Theheterologous polynucleotide can be either stably integrated into thegenome, or can be extra-chromosomal. Preferably, the polynucleotide ofthe present invention is stably integrated into the genome such that thepolynucleotide is passed on to successive generations. A plant cell,tissue, organ, or plant into which the heterologous polynucleotides havebeen introduced is considered “transformed”, “transfected”, or“transgenic”. Direct and indirect progeny of transformed plants or plantcells that also contain the heterologous polynucleotide are alsoconsidered transgenic.

Identification of Plants with an Improved Pathogen Resistance Phenotype

We used an Arabidopsis activation tagging screen to identify theassociation between the gene we have designated “PPR1 (for P. parasiticaResistant),” predicted to encode a protein Ethylene Response Factor 1(ERF1)-like protein, and an altered pathogen resistance phenotype,specifically, increased resistance to the fungal pathogen P. parasitica(downy mildew), a biotrophic pathogen controlled by the salicylic-acid(SA) dependent resistance pathway. Briefly, and as further described inthe Examples, a large number of Arabidopsis plants were mutated with thepSKI015 vector, which comprises a T-DNA from the Ti plasmid ofAgrobacterium tumifaciens, a viral enhancer element, and a selectablemarker gene (Weigel et al, supra). When the T-DNA inserts into thegenome of transformed plants, the enhancer element can causeup-regulation genes in the vicinity, generally within about 10 kilobase(kb) of the insertion. T1 plants were exposed to the selective agent inorder to specifically recover transformed plants that expressed theselectable marker and therefore harbored T-DNA insertions. Samples ofapproximately 18 T2 seed were planted, grown to seedlings, andinoculated with P. parasitica spores. Disease symptoms on individualplants were scored based on the number of conidiophores that emerged.Accordingly, plants on which growth of conidiophores was reduced wereidentified as pathogen resistant.

An Arabidopsis line that showed increased resistance to P. parasiticainfection was identified. The association of the PPR1 gene with thepathogen resistance phenotype was discovered by analysis of the genomicDNA sequence flanking the T-DNA insertion in the identified line.Accordingly, PPR1 genes and/or polypeptides may be employed in thedevelopment of genetically modified plants having a modified pathogenresistance phenotype. PPR1 genes may be used in the generation of cropsand/or other plant species that have improved resistance to infection byP. parasitica and other oomycetes and may also be useful the generationof plant with improved resistance to fungal, bacterial, and/or otherpathogens. Mis-expression of PPR1 genes may thus reduce the need forfungicides and/or pesticides. The modified pathogen resistance phenotypemay further enhance the overall health of the plant.

PPRI Nucleic Acids and Polypeptides

Arabidopsis PPR1 nucleic acid (coding) sequence is provided in SEQ IDNO:1 and in Genbank entry GI 7363407, nucleotides 8077-8811. Thecorresponding protein sequence is provided in SEQ ID NO:2 and in GI8844121.

As used herein, the term “PPR1 polypeptide” refers to a full-length PPR1protein or a fragment, derivative (variant), or ortholog thereof that is“functionally active,” meaning that the protein fragment, derivative, orortholog exhibits one or more or the functional activities associatedwith the polypeptide of SEQ ID NO:2. In one preferred embodiment, afunctionally active PPR1 polypeptide causes an altered pathogenresistance phenotype when mis-expressed in a plant. In a furtherpreferred embodiment, misexpression of the functionally active PPR1polypeptide causes increased resistance to P. parasitica and/or otheroomycetes. In another embodiment, a functionally active PPR1 polypeptideis capable of rescuing defective (including deficient) endogenous PPR1activity when expressed in a plant or in plant cells; the rescuingpolypeptide may be from the same or from a different species as thatwith defective activity. In another embodiment, a functionally activefragment of a full length PPR1 polypeptide (i.e., a native polypeptidehaving the sequence of SEQ ID NO:2 or a naturally occurring orthologthereof) retains one of more of the biological properties associatedwith the full-length PPR1 polypeptide, such as signaling activity,binding activity, catalytic activity, or cellular or extra-cellularlocalizing activity. Some preferred PPR1 polypeptides display DNAbinding activity. A PPR1 fragment preferably comprises a PPR1 domain,such as a C- or N-terminal or catalytic domain, among others, andpreferably comprises at least 10, preferably at least 20, morepreferably at least 25, and most preferably at least 50 contiguous aminoacids of a PPR1 protein. Functional domains can be identified using thePFAM program (Bateman et al. 1999 Nucleic Acids Res 27:260-262; websiteat pfam.wustl.edu). A preferred PPR1 fragment comprises an AP2 domain(PF00847). In SEQ ID NO:2, the AP2 domain is located at approximatelyamino acids 79-144. Functionally active variants of full-length PPR1polypeptides or fragments thereof include polypeptides with amino acidinsertions, deletions, or substitutions that retain one of more of thebiological properties associated with the full-length PPR1 polypeptide.In some cases, variants are generated that change the post-translationalprocessing of a PPR1 polypeptide. For instance, variants may havealtered protein transport or protein localization characteristics oraltered protein half-life compared to the native polypeptide.

As used herein, the term “PPR1 nucleic acid” encompasses nucleic acidswith the sequence provided in or complementary to the sequence providedin SEQ ID NO:1, as well as functionally active fragments, derivatives,or orthologs thereof. A PPR1 nucleic acid of this invention may be DNA,derived from genomic DNA or cDNA, or RNA.

In one embodiment, a functionally active PPR1 nucleic acid encodes or iscomplementary to a nucleic acid that encodes a functionally active PPR1polypeptide. Included within this definition is genomic DNA that servesas a template for a primary RNA transcript (i.e., an mRNA precursor)that requires processing, such as splicing, before encoding thefunctionally active PPR1 polypeptide. A PPR1 nucleic acid can includeother non-coding sequences, which may or may not be transcribed; suchsequences include 5′ and 3′ UTRs, polyadenylation signals and regulatorysequences that control gene expression, among others, as are known inthe art. Some polypeptides require processing events, such asproteolytic cleavage, covalent modification, etc., in order to becomefully active. Accordingly, functionally active nucleic acids may encodethe mature or the pre-processed PPR1 polypeptide, or an intermediateform. A PPR1 polynucleotide can also include heterologous codingsequences, for example, sequences that encode a marker included tofacilitate the purification of the fused polypeptide, or atransformation marker.

In another embodiment, a functionally active PPR1 nucleic acid iscapable of being used in the generation of loss-of-function pathogenresistance phenotypes, for instance, via antisense suppression,co-suppression, etc.

In one preferred embodiment, a PPR1 nucleic acid used in the methods ofthis invention comprises a nucleic acid sequence that encodes or iscomplementary to a sequence that encodes a PPR1 polypeptide having atleast 50%, 60%, 70%, 75%, 80%, 85%, 90%, 95% or more sequence identityto the polypeptide sequence presented in SEQ ID NO:2.

In another embodiment a PPR1 polypeptide of the invention comprises apolypeptide sequence with at least 50% or 60% identity to the PPR1polypeptide sequence of SEQ ID NO:2, and may have at least 70%, 80%,85%, 90% or 95% or more sequence identity to the PPR1 polypeptidesequence of SEQ ID NO:2. In another embodiment, a PPR1 polypeptidecomprises a polypeptide sequence with at least 50%, 60%, 70%, 80%, 85%,90% or 95% or more sequence identity to a functionally active fragmentof the polypeptide presented in SEQ ID NO:2, such as an AP2 domain. Inyet another embodiment, a PPR1 polypeptide comprises a polypeptidesequence with at least 50%, 60%, 70%, 80%, or 90% identity to thepolypeptide sequence of SEQ ID NO:2 over its entire length and comprisesan AP2 domain.

In another aspect, a PPR1 polynucleotide sequence is at least 50% to 60%identical over its entire length to the PPR1 nucleic acid sequencepresented as SEQ ID NO:1, or nucleic acid sequences that arecomplementary to such a PPR1 sequence, and may comprise at least 70%,80%, 85%, 90% or 95% or more sequence identity to the PPR1 sequencepresented as SEQ ID NO: 1 or a functionally active fragment thereof, orcomplementary sequences.

As used herein, “percent (%) sequence identity” with respect to aspecified subject sequence, or a specified portion thereof, is definedas the percentage of nucleotides or amino acids in the candidatederivative sequence identical with the nucleotides or amino acids in thesubject sequence (or specified portion thereof), after aligning thesequences and introducing gaps, if necessary to achieve the maximumpercent sequence identity, as generated by the program WU-BLAST-2.0a19(Altschul et al. 1990, J. Mol. Biol. 215:403-410; website atblast.wustl.edu/blast/README.html) with search parameters set to defaultvalues. The HSP S and HSP S2 parameters are dynamic values and areestablished by the program itself depending upon the composition of theparticular sequence and composition of the particular database againstwhich the sequence of interest is being searched. A “% identity value”is determined by the number of matching identical nucleotides or aminoacids divided by the sequence length for which the percent identity isbeing reported. “Percent (%) amino acid sequence similarity” isdetermined by doing the same calculation as for determining % amino acidsequence identity, but including conservative amino acid substitutionsin addition to identical amino acids in the computation. A conservativeamino acid substitution is one in which an amino acid is substituted foranother amino acid having similar properties such that the folding oractivity of the protein is not significantly affected. Aromatic aminoacids that can be substituted for each other are phenylalanine,tryptophan, and tyrosine; interchangeable hydrophobic amino acids areleucine, isoleucine, methionine, and valine; interchangeable polar aminoacids are glutamine and asparagine; interchangeable basic amino acidsare arginine, lysine and histidine; interchangeable acidic amino acidsare aspartic acid and glutamic acid; and interchangeable small aminoacids are alanine, serine, threonine, cysteine and glycine.

Derivative nucleic acid molecules of the subject nucleic acid moleculesinclude sequences that hybridize to the nucleic acid sequence of SEQ IDNO:1. The stringency of hybridization can be controlled by temperature,ionic strength, pH, and the presence of denaturing agents such asformamide during hybridization and washing. Conditions routinely usedare well known (see, e.g., Current Protocol in Molecular Biology, Vol.1, Chap. 2.10, John Wiley & Sons, Publishers (1994); Sambrook et al.,supra). In some embodiments, a nucleic acid molecule of the invention iscapable of hybridizing to a nucleic acid molecule containing thenucleotide sequence of SEQ ID NO:1 under stringent hybridizationconditions that comprise: prehybridization of filters containing nucleicacid for 8 hours to overnight at 65° C. in a solution comprising 6×single strength citrate (SSC) (1×SSC is 0.15 M NaCl, 0.015 M Na citrate;pH 7.0), 5×Denhardt's solution, 0.05% sodium pyrophosphate and 100 μg/mlherring sperm DNA; hybridization for 18-20 hours at 65° C. in a solutioncontaining 6×SSC, 1×Denhardt's solution, 100 μg/ml yeast tRNA and 0.05%sodium pyrophosphate; and washing of filters at 65° C. for 1 hour in asolution containing 0.2×SSC and 0.1% SDS (sodium dodecyl sulfate). Inother embodiments, moderately stringent hybridization conditions areused that comprise: pretreatment of filters containing nucleic acid for6 hours at 40° C. in a solution containing 35% formamide, 5×SSC, 50 mMTris-HCl (pH 7.5), 5 mM EDTA, 0.1% PVP, 0.1% Ficoll, 1% BSA, and 500μg/ml denatured salmon sperm DNA; hybridization for 18-20 hours at 40°C. in a solution containing 35% formamide, 5×SSC, 50 mM Tris-HCl (pH7.5), 5 mM EDTA, 0.02% PVP, 0.02% Ficoll, 0.2% BSA, 100 μg/ml salmonsperm DNA, and 10% (wt/vol) dextran sulfate; followed by washing twicefor 1 hour at 55° C. in a solution containing 2×SSC and 0.1% SDS.Alternatively, low stringency conditions can be used that comprise:incubation for 8 hours to overnight at 37° C. in a solution comprising20% formamide, 5×SSC, 50 mM sodium phosphate (pH 7.6), 5×Denhardt'ssolution, 10% dextran sulfate, and 20 μg/ml denatured sheared salmonsperm DNA; hybridization in the same buffer for 18 to 20 hours; andwashing of filters in 1×SSC at about 37° C. for 1 hour.

As a result of the degeneracy of the genetic code, a number ofpolynucleotide sequences encoding a PPR1 polypeptide can be produced.For example, codons may be selected to increase the rate at whichexpression of the polypeptide occurs in a particular host species, inaccordance with the optimum codon usage dictated by the particular hostorganism (see, e.g., Nakamura et al 1999 Nucleic Acids Res 27:292). Suchsequence variants may be used in the methods of this invention.

The methods of the invention may use orthologs of the Arabidopsis PPR1.Methods of identifying the orthologs in other plant species are known inthe art. Normally, orthologs in different species retain the samefunction, due to presence of one or more protein motifs and/or3-dimensional structures. In evolution, when a gene duplication eventfollows speciation, a single gene in one species, such as Arabidopsis,may correspond to multiple genes (paralogs) in another. As used herein,the term “orthologs” encompasses paralogs. When sequence data isavailable for a particular plant species, orthologs are generallyidentified by sequence homology analysis, such as BLAST analysis,usually using protein bait sequences. Sequences are assigned as apotential ortholog if the best hit sequence from the forward BLASTresult retrieves the original query sequence in the reverse BLAST(Huynen and Bork 1998, Proc Natl Acad Sci 95:5849-5856; Huynen et al.2000, Genome Research 10: 1204-1210). Programs for multiple sequencealignment, such as CLUSTAL (Thompson et al. 1994 Nucleic Acids Res22:4673-4680) may be used to highlight conserved regions and/or residuesof orthologous proteins and to generate phylogenetic trees. In aphylogenetic tree representing multiple homologous sequences fromdiverse species (e.g., retrieved through BLAST analysis), orthologoussequences from two species generally appear closest on the tree withrespect to all other sequences from these two species. Structuralthreading or other analysis of protein folding (e.g., using software byProCeryon, Biosciences, Salzburg, Austria) may also identify potentialorthologs. Nucleic acid hybridization methods may also be used to findorthologous genes and are preferred when sequence data are notavailable. Degenerate PCR and screening of cDNA or genomic DNA librariesare common methods for finding related gene sequences and are well knownin the art (see, e.g., Sambrook, supra; Dieffenbach and Dveksler (Eds.)PCR Primer: A Laboratory Manual, Cold Spring Harbor Laboratory Press,NY, 1989). For instance, methods for generating a cDNA library from theplant species of interest and probing the library with partiallyhomologous gene probes are described in Sambrook et al. A highlyconserved portion of the Arabidopsis PPR1 coding sequence may be used asa probe. PPR1 ortholog nucleic acids may hybridize to the nucleic acidof SEQ ID NO:1 under high, moderate, or low stringency conditions. Afteramplification or isolation of a segment of a putative ortholog, thatsegment may be cloned and sequenced by standard techniques and utilizedas a probe to isolate a complete cDNA or genomic clone. Alternatively,it is possible to initiate an EST project to generate a database ofsequence information for the plant species of interest. In anotherapproach, antibodies that specifically bind known PPR1 polypeptides areused for ortholog isolation. Western blot analysis can determine that aPPR1 ortholog (i.e., an orthologous protein) is present in a crudeextract of a particular plant species. When reactivity is observed, thesequence encoding the candidate ortholog may be isolated by screeningexpression libraries representing the particular plant species.Expression libraries can be constructed in a variety of commerciallyavailable vectors, including lambda gt11, as described in Sambrook, etal., supra. Once the candidate ortholog(s) are identified by any ofthese means, candidate orthologous sequence are used as bait (the“query”) for the reverse BLAST against sequences from Arabidopsis orother species in which PPR1 nucleic acid and/or polypeptide sequenceshave been identified.

PPR1 nucleic acids and polypeptides may be obtained using any availablemethod. For instance, techniques for isolating cDNA or genomic DNAsequences of interest by screening DNA libraries or by using polymerasechain reaction (PCR), as previously described, are well known in theart. Alternatively, nucleic acid sequence may be synthesized. Any knownmethod, such as site directed mutagenesis (Kunkel et al. 1991, MethodsEnzymol 204:125-39), may be used to introduce desired changes into acloned nucleic acid.

In general, the methods of the invention involve incorporating thedesired form of the PPR1 nucleic acid into a plant expression vector fortransformation of in plant cells, and the PPR1 polypeptide is expressedin the host plant.

An isolated PPR1 nucleic acid molecule is other than in the form orsetting in which it is found in nature and is identified and separatedfrom least one contaminant nucleic acid molecule with which it isordinarily associated in the natural source of the PPR1 nucleic acid.However, an isolated PPR1 nucleic acid molecule includes PPR1 nucleicacid molecules contained in cells that ordinarily express PPR1 where,for example, the nucleic acid molecule is in a chromosomal locationdifferent from that of natural cells.

Generation of Genetically Modified Plants with a Pathogen ResistancePhenotype

PPR1 nucleic acids and polypeptides may be used in the generation ofgenetically modified plants having a modified pathogen resistancephenotype; in general, improved resistance phenotypes are of interest.Pathogenic infection may affect seeds, fruits, blossoms, foliage, stems,tubers, roots, etc. Accordingly, resistance may be observed in any partof the plant. In a preferred embodiment, altered expression of the PPR1gene in a plant is used to generate plants with increased resistance toP. parasitica. In a further preferred embodiment, plants thatmis-express PPR1 may also display altered resistance to other pathogens.Other oomycete pathogens of interest include Pythium spp, Phytophthoraspp, Bremia lactucae, Peronosclerospora spp., Pseudoperonospora,Sclerophthora macrospora, Sclerospora graminicola, Plasmopara viticola,and Albugo candidia. Fungal pathogens of interest include Alternariabrassicicola, Botrytis cinerea, Erysiphe cichoracearum, Fusariumoxysporum, Plasmodiophora brassica, Rhizoctonia solani, Colletotrichumcoccode, Sclerotinia spp., Aspergillus spp., Penicillium spp., Ustilagospp., and Tilletia spp. Bacterial pathogens of interest includeAgrobacterium tumefaciens, Erwinia tracheiphila, Erwinia stewartii,Xanthomonas phaseoli, Erwinia aniylovora, Erwinia carotovora,Pseudomonas syringae, Pelargonium spp, Pseudomonas cichorii, Xanihomonasfragariae, Pseudomonas morsprunorum, Xanthomonas campestris.

The methods described herein are generally applicable to all plants.Although activation tagging and gene identification is carried out inArabidopsis, the PPR1 gene (or an ortholog, variant or fragment thereof)may be expressed in any type of plant. In preferred embodiments, theinvention is directed to crops including maize, soybean, cotton, rice,wheat, barley, tomato, canola, turfgrass, and flax. Other crops includealfalfa, tobacco, and other forage crops. The invention may also bedirected to fruit- and vegetable-bearing plants, plants used in the cutflower industry, grain-producing plants, oil-producing plants, andnut-producing plants, among others.

The skilled artisan will recognize that a wide variety of transformationtechniques exist in the art, and new techniques are continually becomingavailable. Any technique that is suitable for the target host plant canbe employed within the scope of the present invention. For example, theconstructs can be introduced in a variety of forms including, but notlimited to as a strand of DNA, in a plasmid, or in an artificialchromosome. The introduction of the constructs into the target plantcells can be accomplished by a variety of techniques, including, but notlimited to Agrobacterium-mediated transformation, electroporation,microinjection, microprojectile bombardment calcium-phosphate-DNAco-precipitation or liposome-mediated transformation of a heterologousnucleic acid. The transformation of the plant is preferably permanent,i.e. by integration of the introduced expression constructs into thehost plant genome, so that the introduced constructs are passed ontosuccessive plant generations. Depending upon the intended use, aheterologous nucleic acid construct comprising a PPR1 polynucleotide mayencode the entire protein or a biologically active portion thereof.

In one embodiment, binary Ti-based vector systems may be used totransfer polynucleotides. Standard Agrobacterium binary vectors areknown to those of skill in the art, and many are commercially available(e.g., pBI121 Clontech Laboratories, Palo Alto, Calif.).

The optimal procedure for transformation of plants with Agrobacteriumvectors will vary with the type of plant being transformed. Exemplarymethods for Agrobacterium-mediated transformation include transformationof explants of hypocotyl, shoot tip, stem or leaf tissue, derived fromsterile seedlings and/or plantlets. Such transformed plants may bereproduced sexually, or by cell or tissue culture. Agrobacteriumtransformation has been previously described for a large number ofdifferent types of plants and methods for such transformation may befound in the scientific literature.

Expression (including transcription and translation) of PPR1 may beregulated with respect to the level of expression, the tissue type(s)where expression takes place and/or developmental stage of expression. Anumber of heterologous regulatory sequences (e.g., promoters andenhancers) are available for controlling the expression of a PPR1nucleic acid. These include constitutive, inducible and regulatablepromoters, as well as promoters and enhancers that control expression ina tissue- or temporal-specific manner. Exemplary constitutive promotersinclude the raspberry E4 promoter (U.S. Pat. Nos. 5,783,393 and5,783,394), the 35S CaMV (Jones et al. 1992 Transgenic Res 1:285-297),the CsVMV promoter (Verdaguer et al. 1998 Plant Mol Biol 37:1055-1067)and the melon actin promoter (published PCT application WO0056863).Exemplary tissue-specific promoters include the tomato E4 and E8promoters (U.S. Pat. No. 5,859,330) and the tomato 2AII gene promoter(Van Haaren et al. 1993 Plant Mol Bio 21:625-640). In one preferredembodiment, PPR1 expression is under the control of a pathogeninduciblepromoter (Rushton et al. 2002 The Plant Cell 14:749-762)

In one preferred embodiment, PPR1 expression is under control ofregulatory sequences from genes whose expression is associated with theCs VMV promoter.

In yet another aspect, in some cases it may be desirable to inhibit theexpression of endogenous PPR1 in a host cell. Exemplary methods forpracticing this aspect of the invention include, but are not limited toantisense suppression (Smith et al. 1988, Nature 334:724-726; van derKrol et al. 1988, Biotechniques 6:958-976); co-suppression (Napoli etal. 1990, Plant Cell 2:279-289); ribozymes (PCT Publication WO97/10328); and combinations of sense and antisense (Waterhouse et al.1998, Proc. Natl. Acad. Sci. USA 95: 13959-13964). Methods for thesuppression of endogenous sequences in a host cell typically employ thetranscription or transcription and translation of at least a portion ofthe sequence to be suppressed. Such sequences may be homologous tocoding as well as non-coding regions of the endogenous sequence.Antisense inhibition may use the entire cDNA sequence (Sheehy et al.1988, Proc. Natl. Acad. Sci. USA 85:8805-8809), a partial cDNA sequenceincluding fragments of 5′ coding sequence, (Cannon et al. 1990, PlantMolec. Biol. 15:39-47), or 3′ non-coding sequences (Ch'ng et al. 1989,Proc. Natl. Acad. Sci. USA 86:10006-10010). Cosuppression techniques mayuse the entire cDNA sequence (Napoli et al., supra; van der Krol et al.1990, The Plant Cell 2:291-299), or a partial cDNA sequence (Smith etal. 1990, Mol. Gen. Genetics 224:477-481).

Standard molecular and genetic tests may be performed to further analyzethe association between a gene and an observed phenotype. Exemplarytechniques are described below.

1. DNA/RNA Analysis

The stage- and tissue-specific gene expression patterns in mutant versuswild-type lines may be determined, for instance, by in situhybridization. Analysis of the methylation status of the gene,especially flanking regulatory regions, may be performed. Other suitabletechniques include overexpression, ectopic expression, expression inother plant species and gene knock-out (reverse genetics, targetedknock-out, viral induced gene silencing [VIGS, see Baulcombe 1999, ArchVirol Suppl 15:189-201]).

In a preferred application expression profiling, generally by microarrayanalysis, is used to simultaneously measure differences or inducedchanges in the expression of many different genes. Techniques for microarray analysis are well known in the art (Schena et al. 1995, Science270:467-470; Baldwin et al. 1999, Cur Opin Plant Biol. 2(2):96-103;Dangond 2000, Physiol Genomics 2:53-58; van Hal et al. 2000 J Biotechnol78:271-280; Richmond and Somerville 2000, Curr Opin Plant Biol 3:108-116). Expression profiling of individual tagged lines may beperformed. Such analysis can identify other genes that are coordinatelyregulated as a consequence of the overexpression of the gene ofinterest, which may help to place an unknown gene in a particularpathway.

2. Gene Product Analysis

Analysis of gene products may include recombinant protein expression,antisera production, immunolocalization, biochemical assays forcatalytic or other activity, analysis of phosphorylation status, andanalysis of interaction with other proteins via yeast two-hybrid assays.

3. Pathway Analysis

Pathway analysis may include placing a gene or gene product within aparticular biochemical, metabolic or signaling pathway based on itsmis-expression phenotype or by sequence homology with related genes.Alternatively, analysis may comprise genetic crosses with wild-typelines and other mutant lines (creating double mutants) to order the genein a pathway, or determining the effect of a mutation on expression ofdownstream “reporter” genes in a pathway.

Generation of Mutated Plants with a Pathogen Resistance Phenotype

The invention further provides a method of identifying plants that havemutations in endogenous PPR1 that confer increased pathogen resistance,and generating pathogenresistant progeny of these plants that are notgenetically modified. In one method, called “TILLING” (for targetinginduced local lesions in genomes), mutations are induced in the seed ofa plant of interest, for example, using EMS treatment. The resultingplants are grown and self-fertilized, and the progeny are used toprepare DNA samples. PPR1-specific PCR are used to identify whether amutated plant has a PPR1 mutation. Plants having PPR1 mutations may thenbe tested for pathogen resistance, or alternatively, plants may betested for pathogen resistance, and then PPR1-specific PCR is used todetermine whether a plant having increased pathogen resistance has amutated PPR1 gene. TILLING can identify mutations that may alter theexpression of specific genes or the activity of proteins encoded bythese genes (see Colbert et al. 2001, Plant Physiol 126:480-484;McCallum et al. 2000, Nature Biotechnology 18:455-457).

In another method, a candidate gene/Quantitative Trait Locus (QTLs)approach can be used in a marker-assisted breeding program to identifyalleles of or mutations in the PPR1 gene or orthologs of PPR1 that mayconfer increased resistance to pathogens (see Foolad et al. 2002,TheorAppl Genet. 104(6-7):945-958; Rothan et al. 2002, Theor Appl Genet105(1):145-159); Dekkers and Hospital 2002, Nat Rev Genet. January3(1):22-32). Thus, in a further aspect of the invention, a PPR1 nucleicacid is used to identify whether a plant having increased pathogenresistance has a mutation in endogenous PPR1 or has a particular allelethat causes the increased pathogen resistance.

While the invention has been described with reference to specificmethods and embodiments, it will be appreciated that variousmodifications and changes may be made without departing from theinvention. All publications cited herein are expressly incorporatedherein by reference for the purpose of describing and disclosingcompositions and methodologies that might be used in connection with theinvention. All cited patents, patent applications, and sequenceinformation in referenced websites and public databases are alsoincorporated by reference.

EXAMPLES Example 1 Generation of Plants with a Pathogen ResistancePhenotype by Transformation with an Activation Tagging Construct

Mutants were generated using the activation tagging “ACTTAG” vector,pSKI015 (GI 6537289; Weigel D et al., supra). Standard methods were usedfor the generation of Arabidopsis transgenic plants, and wereessentially as described in published application PCT WO0183697.Briefly, T0 Arabidopsis (Col-0) plants were transformed withAgrobacterium carrying the pSKI015 vector, which comprises T-DNA derivedfrom the Agrobacterium Ti plasmid, an herbicide resistance selectablemarker gene, and the 4× CaMV 35S enhancer element. Transgenic plantswere selected at the T1 generation based on herbicide resistance. T2seed was collected from T1 plants and stored in an indexed collection,and a portion of the T2 seed was accessed for the screen.

Approximately 18 T2 seeds from each of the greater than 40,00 linestested were planted in soil. The seed were stratified for three days andthen grown in the greenhouse for seven days. The seedlings wereinoculated with approximately 1×10⁵ conidia per ml P. parasitica sporesand incubated in a dew room at 18° C. and 100% humidity for 24 hours.The plants were then moved to a growth room at 20° C. and 60% relativehumidity with ten-hour long light period for six days. Individual plantswere evaluated for the presence or absence of conidiophores oncotyledons. Lines in which at least a single plant showed noconidiophore growth were re-tested in a secondary screen by releasingthree sets of 18 seed and screening for resistance to P. parasiticagrowth as before.

Lines in which a significant number of plants showed no conidiophoresafter infection were subjected to a tertiary screen. Approximately 54 T2seed were released, planted individually and infected with P. parasiticaas before. The plants were evaluated for the number of conidiophoresgrowing on a single cotyledon and ranked by the following scoringsystem: a score of 0 indicates 0 conidiophores per cotyledon, 1indicates 1-5 conidiophores per cotyledon, 2 indicates 6-10conidiophores per cotyledon, 3 indicates 11-20 conidiophores percotyledon, and 4 indicates greater than 20 conidiophores per cotyledon.

The ACTTAG line designated WO00030248 was identified as having anincreased resistance phenotype. Specifically, 8.8% of individual plantsshowed no conidiophores in the secondary screen. In the tertiary screen,7 plants scored as 0, 14 scored 1, 20 scored 2, 9 scored 3 and 2 scored4. Control (wild-type Col-0) plants displayed significantly greatersusceptibility; 0 plants scored 0, 1 plant scored 1, 1 plant scored 2, 6plants scored 3, and 9 plants scored 4.

Plants from line WO00030248 also displayed altered morphologicalphenotypes. In the T1 generation, these plants displayed leaf petioleand leaf epidermis phenotypes. In the T2 generation, these plantsdisplayed leaf petiole and leaf epidermis phenotypes, as well as lateflowering and reduced size.

The insertion mutation was predicted to have a dominant or semi-dominanteffect. Gentoyping of individual WO00030248 T2 plants analyzed in thetertiary screen indicated that plants that were homozygous for theinsert were more resistant to P. parasitica infection than wereheterozygotes, which were more resistance than wild-type plants.

The dominant P. parasitica resistance phenotype in WO00030248 isheritable. Approximately 54 individual WO00030248 plants from two T3families were analyzed for resistance to P. parasitica. The resultsindicated that plants homozygous or heterozygous for the insert showedcomparable resistance to infection by P. parasitica.

Example 2 Characterization of the T-DNA Insertion in Plants Exhibitingthe Altered Pathogen Resistance Phenotype

We performed standard molecular analyses, essentially as described inpatent application PCT WO0183697, to determine the site of the T-DNAinsertion associated with the increased pathogen resistance phenotype.Briefly, genomic DNA was extracted from plants exhibiting increasedpathogen resistance. PCR, using primers specific to the pSKI015 vector,confirmed the presence of the 35S enhancer in plants from lineWO00030248, and Southern blot analysis verified the genomic integrationof the ACTTAG T-DNA and showed the presence of a single T-DNA insertionin the transgenic line.

Plasmid rescue was used to recover genomic DNA flanking the T-DNAinsertion, which was then subjected to sequence analysis.

The sequence flanking the left T-DNA border was subjected to a basicBLASTN search and/or a search of the Arabidopsis Information Resource(TAIR) database (available at the arabidopsis.org website), whichrevealed sequence identity to BAC clone F9P14 (GI 7363407), mapped tochromosome 1. The T-DNA inserted at nucleotide 2971 of F9P14. Sequenceanalysis revealed that the T-DNA had inserted in the vicinity (i.e.,within about 10 kb) of the gene whose nucleotide sequence is presentedas SEQ ID NO: 1 and GI 7363407, nucleotides 8077-8811, and which wedesignated PPR1. Specifically, the TDNA inserted approximately 5 kb 5′to the coding sequence of the PPR1 gene.

Example 3 Analysis of Arabidopsis PPR1 Sequence

The amino acid sequence predicted from the PPR1 nucleic acid sequence ispresented in SEQ ID NO:2 and GI 8844121. PFAM analysis identified an AP2DNA binding domain located at amino acids 79-144. A serine-rich regionis located near the carboxy terminus.

Sequence analyses were performed with BLAST (Altschul et al., supra) andPFAM (Bateman et al., supra), among others. BLAST analysis indicatedthat SEQ ID NO:2 has similarity to the DNA binding protein S25-XP1 fromNicotiana tabacum (GI 7489116 and GI 1732406), the Arabidopsis EthyleneResponse Factor (ERF1; GI 4128210, GI 4128208, and GI 15229405), aputative DNA binding protein from Oryza sativa (GI 19034045), anethylene response factor ERF1-like protein from Oryza sativa (GI24060083), and transcription factor TSRF1 from Lycopersicon esculentum(GI 23452024). The top BLAST hit was GI 22326027 (At2g31230; SEQ IDNO:3), which is annotated as “ethylene response factor, putative”, andshares 70% overall identity with PPR1 (SEQ ID NO:2). GI 22326027, likePPR1, has an AP2 domain and has a serine-rich region near the carboxyterminus. ERF1 lacks a similar serine-rich region. GI 22326027 alsoshares high identity with PPR1 at the carboxy terminal 32 amino acids(approx. 73% identity). By comparison, ERF1 shares only about 33%identity with PPR1 in this region. Thus, orthologs of PPR1 may beexpected to contain serine-rich regions and share greater sequenceidentity with PPR1 (SEQ ID NO:2) compared to ERF1 (GI 4128210).

Example 4 Confirmation of Phenotype/Genotype Association

RT-PCR analysis showed that the PPR1 gene was overexpressed inpathogenresistant plants from line WO00030248. Specifically, RNA wasextracted from tissues derived from plants exhibiting the pathogenresistance phenotype and from wild type COL-0 plants. RT-PCR wasperformed using primers specific to the sequence presented as SEQ IDNO:1, to other predicted genes in the vicinity of the T-DNA insertion,and to a constitutively expressed actin gene (positive control). Theresults showed that plants displaying the pathogen resistance phenotypeover-expressed the mRNA for the PPR1 gene, indicating the enhancedexpression of the PPR1 gene is correlated with the pathogen resistancephenotype.

Example 5 Recapitulation of Pathogen Resistance Phenotype

Arabidopsis plants of the Ws ecotype were transformed by agrobacteriummediated transformation with a construct containing the coding sequencesof the PPR1 gene (Atlg06160, gil 15221402) behind the CsVMV promoter andin front of the nos terminator or a control gene unrelated to pathogenresistance. Both of these constructs contain the nptII gene to conferkanamycin resistance in plants. T1 seed was harvested from thetransformed plants and transformants selected by germinating seed onagar medium containing kanamycin. Kanamycin resistant transformants weretransplanted to soil after 7 days and grown for 4 weeks. Control plantswere germinated on agar medium without kanamycin, transplanted to soilafter 7 days and grown in soil for 4 weeks.

To evaluate pathogen resistance, transformants and control plants weresprayed with a suspension of 1×10⁵ conidia per ml of P. parasitica,incubated at 100% humidity for 1 day, and grown for 6 more days in thegrowth room. After this growth period, plants were rated for severity ofdisease symptoms. A score of 0 means the leaves had 0-10% of the numberof conidiophores growing on the leaf surface as a fully susceptibleplant, 1 means 10-25% the number of conidiophores, 2 means 25-50%, 3means 50-75% and 4 means 75-100%. Fifty-two plants transformed withPPR1, 50 plants transformed with the control gene and 10 control plantswere examined.

Degree-of-infection scores were obtained from each plant tested. As agroup, the PPR1 transformants were more resistant to P. parasiticainfection than control plants. In PPR1 transformants, 11.5% were scoredas 0, 15.4% as 1, 21.5% as 2, 30.8% as 3 and 21.5% as 4. In plantstransformed with the control gene, only 4% scored as 0, 4% as 1, 6% as2, 4% as 3, while 82% scored as 4. In control plants, 0% scored 0, 1,and 2, 10% scored 3 and 90% scored 4. These data show that plantsover-expressing PPR1 are significantly more resistant to P. parasiticainfection than wild-type plants.

Further analysis of plants constitutively expressing PPR1, showed thatthey constitutively express endogenous PDF1.2, a pathogenesis related(PR) protein that is a molecular marker for the jasmonic acid(JA)-dependent resistance pathway. The JA-dependent resistance pathwaycontrols necrotrophic fungi and oomycetes such as Alemaria brassicola orBotrytis cinerea. These plants also constitutively express endogenousPR1, a marker for the salicylic acid (SA)-dependent resistance pathway.The SA-dependent resistance pathway controls bacterial pathogens such asPseudomonas spp. Xanthomonas spp., and Erwinia and biotrophic fungi andoomycetes such as Erysiphe cichoracearum and Peronospora parasitica.Thus, plants genetically modified to overexpress a PPR1 ortholog, may besimilarly expected to also overexpress endogenous PR1 and PDF1.2relative to non-transgenic plants and be resistant to pathogens that arecontrolled by both the SA- and JA-dependent resistance pathways. Incontrast, plants that constitutively express ERF1 constitutively expressPDF1.2 but not PR1 and are resistant to pathogens controlled by only theJA-dependent resistance pathway (Berrocal-Lobo et al. 2002, The PlantJournal 29(1):23-32).

1. A method of producing increased pathogen resistance in a plant, saidmethod comprising: introducing into progenitor cells of the plant aplant transformation vector comprising a nucleotide sequence thatencodes SEQ ID NO:2 or a polypeptide having at least 95% sequenceidentity with SEQ ID NO:2, and growing the transformed progenitor cellsto produce a transgenic plant, wherein said transgenic plant exhibitsconstitutive expression of endogenous PR1 protein and said increasedresistance to pathogens controlled by the salicylic acid-dependentresistance pathway relative to a non-transgenic plant.
 2. A transgenicplant produced by the method of claim
 2. 3. A transgenic plant partobtained from the plant according to claim
 2. 4. A transgenic plantcomprising a plant transformation vector comprising a nucleotidesequence encoding a PPR1 polypeptide having at least 95% sequenceidentity with SEQ ID NO:2, wherein said transgenic plant exhibitsconstitutive expression of endogenous PPR1 protein and has increasedresistance to a pathogen controlled by salicylic acid-dependentresistance pathway relative to a control plant.
 5. The transgenic plantof claim 4, wherein the PPR1 polypeptide is SEQ ID NO:2.